Treatment of Vocal Cords With Autologous Dermal Fibroblast Formulation

ABSTRACT

Dosage units consist of an autologous cell therapy product composed of fibroblasts grown for each individual to be treated for augmentation or regeneration of vocal cords. The suspension of autologous fibroblasts, grown from a biopsy of each individual&#39;s own buccal mucosa or skin using current good manufacturing practices (CGMP) and standard tissue culture procedures, is supplied in vials containing cryopreserved fibroblasts or precursors thereof, having a purity of at least 98% fibroblasts and a viability of at least 85%, for administration of from one to six mL, preferably two mL administered three tunes approximately three to six weeks apart, of cells at a concentration of from 1.0-2.0×10 7  cells/mL.

FIELD OF THE INVENTION

This relates to treatment of vocal fold scarring and vocal cordaugmentation using isolated, prepared autologous dermal fibroblasts forinjection in human subjects.

BACKGROUND OF THE INVENTION

The vocal folds of the human larynx are highly specialized structuresthat are capable of self-sustained oscillation for production of soundfor speech, communication, and singing. The vocal folds are dividedanatomically into three tissue layers (Hirano M. Otologia (Fukuoka)1975; 21:239-442). The superficial layer is the vocal fold epithelium,followed by the middle lamina propria layer, and the deep muscular layer(FIG. 1). The epithelial layer is very thin compared to the other layersand acts biomechanically as a functional unit with the lamina proprialayer and thus these two layers are combined and called the “cover”layer in biomechanical studies of the vocal fold. The lamina proprialayer is an amorphous, paucicellular layer composed mostly offibroblasts, macrophages and extracellular matrix molecules (Gray et al.Laryngoscope 1999; 109:845-54). This layer provides the appropriateviscoelasticity (mucosal pliability) for normal oscillation of the vocalfolds during phonation which can be appreciated clinically as mucosalwaves on the vocal fold surface upon videostroboscopic examination ofthe larynx (Hirano et al. J Voice 2009; 23(4):399-407). Vocal foldscarring is a pathologic condition that results from loss of the laminapropria layer and leads to glottic insufficiency and diminished orabsent mucosal waves on the vocal fold surface, and is a common clinicalproblem resulting in dysphonia. Treatment of lamina propria loss is ofspecial interest because there is currently no effective replacementtherapy.

The most frequent etiology for vocal fold lamina propria loss is surgeryon the vocal fold for benign and malignant disorders. Other causesinclude a variety of traumatic, neoplastic, iatrogenic, inflammatory,and idiopathic disorders (Rosen Otolaryngol Clin North Am 2000;33:1081-6). The resulting voice is often rough and breathy, of poorquality, and perceived by patients as a severe communication handicap.Histologically, lamina propria loss after iatrogenic vocal fold injuryoccurs as the layer is replaced with dense and disorganized collagendeposits (Hirano Curr Opin Otolaryngol Head Neck Surg. 2005 June;13(3):143-7). This increased fibrosis is referred to clinically as vocalfold scar.

Vocal fold scar is a challenging problem for the otolaryngologistbecause effective therapy for this condition is currently lacking andrehabilitation of patients is difficult. Management of vocal fold scarswith autologous fat implantation and autologous fascia augmentation hasbeen reported but treatment results have been less than satisfactory(Benninger et al. Otolaryngol Head Neck Surg 1996; 115:474-82;Neuenschwander et al. J Voice 2001; 15:295-304; Duke et al. Laryngoscope2001; 111:759-64). This is because currently no biomaterial exists thatmatches the native viscoelastic properties of this layer.

There are currently two potential therapeutic modalities for vocal foldscars: (a) Injection of biomaterials into the lamina propriacompartment, and (b) cellular based therapy (Hsiung et al. Laryngoscope2000; 110:1026-33; Chhetri et al. Otolaryngol Head Neck Surg 2004; 131:864-70). The biomaterial approach has been problematic because durablebiomaterials that also have the appropriate viscoelastic properties haveyet to be developed. Current biomaterials are all too stiff to beinjected into the lamina propria compartment.

The ability to take cells from an individual, expand those cells in thelaboratory, and inject them into the same individual to repairsymptomatic tissue defects is a newly evolving therapeutic modality inmedicine. Initial work in this area of “cultured autologous cellular”therapy was directed towards chondrocytcs. A Federal Drug Administration(FDA) approved autologous cultured chondrocyte product (Carticel®,Genzymebiosurgery, Cambridge, Mass.) has been available since 1997 fororthopedic use (Hayflick L and Moorhead P S. Exp Cell Research 1961;25:585-621). Autologous cellular therapy has also been appliedclinically to include transplantation of autologous cultured melanocytesfor treatment of segmental vitiligo (Treco D A et al. Fibroblast cellbiology and gene therapy. In: Chang P L, ed. Somatic Gene Therapy. BocaRaton: CRC Press, 1995:49-60), autologous keratinocytes for treatment ofulcers and burns (Jacobson et al. Am J Speech Lang Pathol 1997; 6:66-70;Hartnick et al. Laryngoscope 2005; 115:4-15), and autologous fibroblastsfor treatment of facial wrinkles (Boss et al. Ann Plast Surg 2000;44:536-42; Watson et al. Arch Facial Plast Surg 1999; 1:165-70; Kanemaruet al Ann Otol Rhinol Laryngol 2003; 112:915-920). No adverse effectssuch as malignant transformation of injected cells or significant tissuereaction have been reported so far with the use of autologous cellulartherapy.

The lamina propria layer is composed primarily of ECM molecules such ascollagen, elastin, and proteoglycans. Fibroblast cells in this layerproduce these ECM molecules (Gray, et al., 1999). Theoretically,injection of autologous fibroblasts into the lamina propria layer couldlead to reconstitution of normal lamina propria ECM components andimprove the voice disorder by re-establishing normal mucosal pliabilityof the vocal folds. Lamina propria replacement therapy must consider thecomplex three dimensional organization of this layer that develops andmatures over a significant time period. At birth, the lamina proprialayer is a hyper-cellular monolayer and it matures over the next sevento thirteen years into the trilaminar layer with the differential fiberand proteoglycan composition seen in adults. This three dimensionalorganization is complex with not only differences in the relativecomposition of the ECM molecules within the layer but also in the threedimensional orientation of collagen and elastic fibers. Lamina propriareplacement therapy therefore needs to address not only theviscoelasticity of the replacement material but its three-dimensionalgeometry and composition as well. Production of such lamina propria withits normal components in proper concentration and configuration remainsa daunting task with currently available tissue engineering techniques.

Fibroblasts are readily obtained from skin or buccal mucosa by punchbiopsy and can be cultured free of other cell types. Human fibroblastsdo not spontaneously become immortal in culture, a property that hassignificant implications when their re-injection into a human being isconsidered. Cultured autologous fibroblast therapy in humans has so farbeen directed mainly in the cosmetic plastic surgery field for thetreatment of facial wrinkles and scars (Boss et al., Ann Plast Surg2000; 44:536-42; Watson et al. Arch Facial Plast Surg 1999; 1:165-70;Kanemaru et al. Ann Otol Rhinol Laryngol 2003; 112:915-920). Boss andcolleagues reported treating 1,000 subjects with cultured autologousfibroblasts. They performed approximately 4,000 injections from 1995through 1999. The follow-up period was 36-48 months. Ninety-two percentof the subjects were satisfied with the therapy. There were a total of13 reported reactions (0.27%) to the injections, of which 11 were mildreactions with redness and swelling that resolved within 48-72 hours.The other two reactions were moderate with swelling and erythema for7-10 days. Watson and colleagues reported a 6-month prospective pilotstudy in 10 adults to assess the efficacy of cultured autologousfibroblasts to treat skin wrinkles and dermal depressions. Microscopicexamination of the injection site was also performed and demonstrated adenser and thicker layer of collagen in the dermal region, absence ofany inflammatory reaction, and viable fibroblasts throughout. No adversereactions were noted clinically or microscopically.

A canine study has previously shown the potential for autologousfibroblast injection therapy for treatment of vocal fold scars. Caninelamina propria replacement therapy was performed where autologousfibroblasts were harvested from buccal mucosal biopsies and expanded inthe laboratory. Fourth or fifth passage fibroblasts were injected intopreviously scarified vocal folds. The scarred vocal folds had absent orseverely limited mucosal waves and poor acoustic parameters. Significantimprovements in mucosal waves and acoustic parameters were obtainedfollowing autologous fibroblasts injection therapy. Chhetri D K et al.Otolaryngol Head Neck Surg 2004; 131: 864-70). Two months later lessatrophy was observed in the treated vocal fold compared to the controlvocal fold. Histologically the injected cells appeared viable. However,phonation studies were not performed and the mucosal wave grade or theacoustic quality of voice was not provided. In another study, humanembryonic stem cells were injected into scarred rabbit vocal folds andhistologic assessment performed one month later (Cedervall, et. al.Laryngoscope 2007; 117:2075-2081). Persistence of the embryonic stemcells was observed and the injected vocal fold was associated withdecreased viscoelasticity (as measured by parallel plate rheometry) ascompared to the untreated but scarred side. Another study performedinjection of autologous fibroblasts from skin into scarred rabbit vocalfolds. In contrast from other reports, this study “primed” thefibroblasts by addition of various factors such as epidermal growthfactor, hepatocyte growth factor (HGF), and decorin to the cell culture(Krishna et al., Otolaryngol Head Neck Surg 2006; 135:937-945). Theyreported that HGF treated cells demonstrated increased synthesis ofhyaluronic acid, and the HGF and decorin treated cells demonstrateddiminished collagen synthesis in vivo.

While demonstration of appropriate changes to the ECM components isimportant, ultimately the resulting vibratory behavior of the vocalfolds is most important. Specifically, the return of vocal fold mucosalpliability as improved mucosal waves upon phonation should be one of theultimate criteria for success. One previous animal study has performedautologous fibroblast injection into scarred vocal folds in a caninemodel and demonstrated return of mucosal waves and improved acousticparameters (Cedervall et al. Laryngoscope 2007; 117:2075-2081). In thatstudy, vocal fold scarring was induced unilaterally in the animals andresulted in absent or severely limited mucosal waves and significantlyworse acoustic parameters. Autologous fibroblasts were harvested fromthe canine buccal mucosa and the cell population was expanded in thelaboratory. Fourth or fifth passage fibroblasts were then injected intopreviously scarified vocal folds. Significant improvements in mucosalwaves and acoustic parameters were obtained after lamina propriareplacement therapy. After therapy, mucosal waves became normal in fouranimals and near normal in the other four. No statistical difference wasfound in mucosal wave grade between baseline and post-therapy. Allanimals tolerated therapy without complications. Histological analysisof the treated vocal folds demonstrated an increased density offibroblasts, collagen, and reticulin, a decreased density of elastin,and no change in hyaluronic acid.

It is therefore an object of the present invention to provide defineddosage unit formulations of autologous dermal fibroblasts for injectioninto human patients for the repair and long term augmentation of vocalcord defects.

It is a further object of the present invention to provide dosage unitformulation that contain stem cells, precursor cells or partiallydifferentiated cells that can be used for the repair and long termaugmentation of vocal cord defects in humans.

SUMMARY OF THE INVENTION

Dosage units consist of an autologous cell therapy product composed offibroblasts grown for each individual to be treated for augmentation orregeneration of vocal cords. The suspension of autologous fibroblasts,grown from a biopsy of each individual's own skin using current goodmanufacturing practices (CGMP) and standard tissue culture procedures,is supplied in vials containing cryopreserved fibroblasts or precursorsthereof, having a purity of at least 98% fibroblasts and a viability ofat least 85%, for administration. In a preferred embodiment, from one tosix mL, for example two mL, is administered three times approximatelythree to six weeks apart. In one embodiment, the cells are at aconcentration of from 1.0-2.0×10⁷ cells/mL. The autologous fibroblastsare thought to increase the synthesis of extracellular matrixcomponents, including collagen. Dosage and timing of administration havebeen demonstrated to be critical to achieving clinically significantoutcomes.

The example describes a clinical study in which five patients wereenrolled with vocal fold scars that ranged from isolated lamina proprialoss to full thickness scarring that included the epithelium. Allsubjects received all treatments and followed up to 12 months.Assessment of safety endpoints showed that the only adverse events weretemporary otalgia during treatment injection in the two patients withfull thickness cover scars. There was no other morbidity or mortality.There were no laboratory abnormalities or other untoward events thatwere considered related to the study treatment. The primary efficacyendpoint of change from baseline in the mucosal wave grade showed asustained improvement from week 8 through the Month 12 visit. A trendfor sustained improvements through month 12 was also noted for the VHI,VAS, and Voice Quality Questionnaire.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the anatomy of the vocal folds from Hirano M.Otologia (Fukuoka) 1975; 21:239-442. The superficial layer is the vocalfold epithelium, followed by the middle lamina propria layer, and thedeep muscular layer.

FIG. 2 is a standardized manufacturing process flow diagram.

FIG. 3 is a graph of VHI score over time (in months).

DETAILED DESCRIPTION OF THE INVENTION I. Autologous FibroblastFormulation

The following definitions are used herein:

ATM Analytical Test Method

AZFICEL-T USAN nomenclature for autologous cultured fibroblastsBULK HARVEST material following final harvest prior to formulation incryopreservation media

CGMP Current Good Manufacturing Practice

CS Cell stack

DMEM Dulbecco's Modification of Eagle's Medium

DMSO Dimethyl sulfoxideDRUG PRODUCT—INJECTION material washed and reformulated inDMEM, vialed and ready for shipment to clinical sitesDRUG SUBSTANCE—CRYOVIAL material formulated in cryopreservation mediaand aliquoted into cryovialsEDTA Ethylenediaminetetra acetic acid

FACS Fluorescence Activated Cell Sorting FBS Fetal Bovine Serum GAGentamicin and Amphotericin B IMDM Iscove's Modified Dulbecco's Medium

IND Investigational New Drug application

PBS Phosphate Buffered Saline PCA Personal Cell Analysis QC QualityControl USP United States Pharmacopeia

A. Sources of Cells

1. Autologous Dermal Fibroblasts

An autologous fibroblast product has been developed. The cell therapyproduct is composed of a suspension of autologous fibroblasts, grownfrom a biopsy of each individual's own skin using standard tissueculture procedures. Skin tissue (dermis and epidermis layers) isbiopsied from a patient's post-auricular area and shipped via next daydelivery to a manufacturing facility at 2-8° C. Fibroblasts isolatedfrom the tissue via enzymatic digestion are expanded to a quantitysufficient for injection into the patient's target treatment area. TheCell therapy product consists of expanded fibroblasts, formulated to thetarget cell concentration and cryopreserved in cryovials, called BulkDrug Substance—Cryovial. The final cell therapy product consists ofthawed Bulk Cell therapy product-Cryovial cells that are thawed, washedand prepared for patient injection.

The cells in the formulation display typical fibroblast morphologieswhen growing in cultured monolayers. Specifically, cells may display anelongated, fusiform or spindle appearance with slender extensions, orcells may appear as larger, flattened stellate cells which may havecytoplasmic leading edges. A mixture of these morphologies may also beobserved. The cells express proteins characteristic of normalfibroblasts including the fibroblast-specific marker, CD90 (Thy-1), a 35kDa cell-surface glycoprotein, and the extracellular matrix protein,collagen.

2. Precursor Cells

The fibroblasts can also be used to create other cell types for tissuerepair or regeneration. Derivation of embryonic stem (ES) cellsgenetically identical to a patient by somatic cell nuclear transfer(SCNT) holds the potential to cure or alleviate the symptoms of manydegenerative diseases while circumventing concerns regarding rejectionby the host immune system. Byrne, et al., Nature 2007 22;450(7169):497-502, used a modified SCNT approach to produce rhesusmacaque blastocysts from adult skin fibroblasts, and successfullyisolated two ES cell lines from these embryos. DNA analysis confirmedthat nuclear DNA was identical to donor somatic cells and thatmitochondrial DNA originated from oocytes. Both cell lines exhibitednormal ES cell morphology, expressed key stem-cell markers, weretranscriptionally similar to control ES cells and differentiated intomultiple cell types in vitro and in vivo. See also Sparman, et al. StemCells. 2009; 27(6):1255-64, Hochedlinger, et al., Development. 2009February; 136(4):509-23 and Kanawaty, et al. Bioessays. 2009 February;31(2):134-8.

Methods are known by which fibroblasts can be de-differentiated intopluripotent cells: cell fusion (Cowan et al. Science. 2005 Aug. 26;309(5739):1369-73), direct reprogramming (Takahashi, et al., Cell. 200730; 131(5):861-72), and somatic cell nuclear transfer (Byrne, et al.2007). Takahashi, et al. demonstrated the generation of iPS cells fromadult human dermal fibroblasts with the same four factors: Oct3/4, Sox2.Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem(ES) cells in morphology, proliferation, surface antigens, geneexpression, epigenetic status of pluripotent cell-specific genes, andtelomerase activity. Furthermore, these cells could differentiate intocell types of the three germ layers in vitro and in teratomas. Thesefindings demonstrate that iPS cells can be generated from adult humanfibroblasts.

B. Preparation of Cells

The autologous fibroblasts in the Drug Substance are derived byenzymatic digest of a biopsy of the recipient's own skin followed byexpansion in culture using standard cell culture techniques. Skin tissue(dermis and epidermis layers) is biopsied from a subject'spost-auricular area. The starting material is composed of three mm punchbuccal mucosa biopsies collected using standard aseptic practices.Buccal mucosa rather than skin biopsy is used as the source forautologous fibroblasts for treatment of vocal cord scarring because thecollagen production profile of buccal mucosa more closesly parallelsthat of the vocal fold fibroblasts as compared to dermal fibroblasts.(Hayflick L and Moorhead P S. Exp Cell Research 1961; 25:585-621; TrecoD A et al. In: Chang P L, ed. Somatic Gene Therapy. Boca Raton: CRCPress, 1995:49-60). Although buccal mucosa is the preferred testlocation, biopsies may be sourced from another location in the mouth,such as hard palette. The biopsies are collected by the treatingphysician, placed into a vial containing sterile phosphate bufferedsaline (PBS). The biopsies are shipped in a 2-8° C. refrigerated shipperback to the manufacturing facility. After arrival at the manufacturingfacility, the biopsy is inspected and, upon acceptance, transferreddirectly to the manufacturing area.

Upon initiation of the process, the biopsy tissue is then washed priorto enzymatic digestion. After washing, a Liberase Digestive EnzymeSolution is added without mincing, and the biopsy tissue is incubated at37.0+2° C. for one hour. Time of biopsy tissue digestion is a criticalprocess parameter that can affect the viability and growth rate of cellsin culture. Liberase is a collagenaseneutral protease enzyme cocktailobtained formulated from Lonza Walkersville, Inc. (Walkersville, Md.)and unformulated from Roche Diagnostics Corp. (Indianapolis, Ind.).

Alternatively, other commercially available collagenases may be used,such as Serva Collagenase NB6 (Helidelburg, Germany). After digestion,initiation Growth Media (IMDM, GA, 10% Fetal Bovine Serum (FBS)) isadded to neutralize the enzyme, cells are pelleted by centrifugation andresuspended in Initiation Growth Media. Alternatively, centrifugation isnot performed, with full inactivation of the enzyme occurring by theaddition of Initiation Growth Media only. Initiation Growth Media isadded prior to seeding of the cell suspension into a T-175 cell cultureflask for initiation of cell growth and expansion. A T-75, T-150. T-185or T-225 flask can be used in place of the T-75 flask. Cells areincubated at 37±2.0° C. with 5.0±1.0% CO₂ and fed with fresh CompleteGrowth Media every three to five days. All feeds in the process areperformed by removing half of the Complete Growth Media and replacingthe same volume with fresh media. Alternatively, full feeds can beperformed. Cells should not remain in the T-175 flask greater than 30days prior to passaging. Confluence is monitored throughout the processto ensure adequate seeding densities during culture splitting.

When cell confluence is greater than or equal to 40% in the T-175 flask,they are trypsinized and seeded into a T-500 flask for continued cellexpansion. Alternately, one or two T-300 flasks, One Layer Cell Stack (1CS), One Layer Cell Factory (1 CF) or a Two Layer Cell Stack (2 CS) canbe used in place of the T-500 Flask. Morphology is evaluated at eachpassage and prior to harvest to monitor the culture purity throughoutthe process. Morphology is evaluated by comparing the observed samplewith visual standards for morphology examination of cell cultures. Thecells display typical fibroblast morphologies when growing in culturedmonolayers. Cells may display either an elongated, fusiform or spindleappearance with slender extensions, or appear as larger, flattenedstellate cells which may have cytoplasmic leading edges. A mixture ofthese morphologies may also be observed. Fibroblasts in less confluentareas can be similarly shaped, but randomly oriented. The presence ofkeratinocytes in cell cultures is also evaluated. Keratinocytes appearround and irregularly shaped and, at higher confluence, they appearorganized in a cobblestone formation. At lower confluence, keratinocytesare observable in small colonies.

Cells are incubated at 37±2.0° C. with 5.0±1.0% CO₂ and fed every threeto five days in the T-500 flask and every five to seven days in the tenlayer cell stack (10 CS). Cells should not remain in the T-500 flask formore than 10 days prior to passaging. QC release testing for safety ofthe Bulk Drug Substance includes sterility and endotoxin testing. Whencell confluence in the T-500 flask is ≦95%, cells are passaged to a 10CS culture vessel. Alternately, two Five Layer Cell Stacks (5 CS) or a10 Layer Cell Factory (10 CF) can be used in place of the 10 CS. Passageto the 10 CS is performed by removing the spent media, washing thecells, and treating with Trypsin-EDTA to release adherent cells in theflasks into the solution. Additional Complete Growth Media is added toneutralize the trypsin and the cells from the T-500 flask are pipettedinto a 2 L bottle containing fresh Complete Growth Media. The contentsof the 2 L bottle are transferred into the 10 CS and seeded across alllayers. Cells are then incubated at 37±2.0° C. with 5.0±1.0% CO₂ and fedwith fresh Complete Growth Media every five to seven days. Cells shouldnot remain in the 10 CS for more than 20 days prior to passaging.

Primary Harvest: When cell confluence in the 10 CS is 95% or more, cellsare harvested. Harvesting is performed by removing the spent media,washing the cells, treating with Trypsin-EDTA to release adherent cellsinto the solution, and adding additional Complete Growth Media toneutralize the trypsin. Cells are collected by centrifugation,resuspended, and in-process Quality Control (QC) testing performed todetermine total viable cell count and cell viability.

If additional cells are required after receiving cell count results fromthe primary 10 CS harvest, an additional passage into multiple cellstacks (up to four 10 CS) is performed (Step 5 a in FIG. 1). Foradditional passaging, cells from the primary harvest are added to a 2 Lmedia bottle containing fresh Complete Growth Media. Resuspended cellsare added to multiple cell stacks and incubated at 37±2.0° C. with5.0±1.0% CO₂. The cell stacks are fed and harvested as described above,except cell confluence must be 80% or higher prior to cell harvest. Theharvest procedure is the same as described for the primary harvestabove. A mycoplasma sample from cells and spent media is collected, andcell count and viability performed as described for the primary harvestabove.

Alternate Manufacturing Methods

Alternatively, cells can be passaged from either the T-175 flask (oralternatives) or the T-500 flask (or alternatives) into a spinner flaskcontaining microcarriers as the cell growth sturface. Microcarriers aresmall bead-like structures that are used as a growth surface foranchorage dependent cells in suspension culture. They are designed toproduce large cell yields in small volumes.

In this apparatus, a volume of Complete Growth Media ranging from 50mL-300 mL is added to a 500 mL, IL or 2 L sterile disposable spinnerflask. Sterile microcarriers are added to the spinner flask. The cultureis allowed to remain static or is placed on a stir plate at a low RPM(15-30 RRM) for a short period of time (1-24 hours) in a 37±2.0° C. with5.0±1.0% CO₂ incubator to allow for adherence of cells to the carriers.After the attachment period, the speed of the spin plate is increased(30-120 RPM). Cells are fed with fresh Complete Growth Media every oneto five days, or when media appears spent by color change.

Cells are collected at regular intervals by sampling the microcarriers,isolating the cells and performing cell count and viability analysis.The concentration of cells per carrier is used to determine when toscale-up the culture. When enough cells are produced, cells are washedwith PBS and harvested from the microcarriers using trypsin-EDTA andseeded back into the spinner flask in a larger amount of microcarriersand higher volume of Complete Growth Media (300 mL-2 L). Alternatively,additional microcarriers and Complete Growth Media can be added directlyto the spinner flask containing the existing microcarrier culture,allowing for direct bead-to-bead transfer of cells without the use oftrypsiziation and reseeding. Alternatively, if enough cells are producedfrom the initial T-175 or T-500 flask, the cells can be directly seededinto the scale-up amount of microcarriers. After the attachment period,the speed of the spin plate is increased (30-120 RPM). Cells are fedwith fresh Complete Growth Media every one to five days, or when mediaappears spent by color change. When the concentration reaches thedesired cell count for the intended indication, the cells are washedwith PBS and harvested using trypsin-EDTA.

Microcarriers used within the disposable spinner flask may be made frompoly blend such as BioNOC II® (Cesco Bioengineering, distributed byBellco Biotechnology, Vineland, N.J.) and FibraCel® (New BrunswickScientific, Edison, N.J.), gelatin, such as Cultispher-G (PercellBiolytica. Astrop, Sweden), cellulose, such as Cytopore™ (GE Healthcare,Piscataway, N.J.) or coated/uncoated polystyrene, such as 2D MicroHex™(Nunc, Weisbaden, Germany), Cytodex® (GE Healthcare, Piscataway, N.J.)or Hy-Q Sphere™ (Thermo Scientific Hyclone, Logan, Utah).

Alternatively, cells can be processed on poly blend 2D microcarrierssuch as BioNOC II® and FibraCel® using an automatic bellow system, suchas FibraStage™ (New Brunswick Scientific, Edison, N.J.) or BelloCell®(Cesco Bioengineering, distributed by Bellco Biotechnology, Vineland,N.J.) in place of the spinner flask apparatus. Cells from the T-175 (oralternatives) or T-500 flask (or alternatives) are passaged into abellow bottle containing microcarriers with the appropriate amount ofComplete Growth Media, and placed into the system. The system pumpsmedia over the microcarriers to feed cells, and draws away media toallow for oxygenation in a repeating fixed cycle. Cells are monitored,fed, washed and harvested in the same sequence as described above.

Alternatively, cells can be processed using automated systems. Afterdigestion of the biopsy tissue or after the first passage is complete(T-175 flask or alternative), cells may be seeded into an automateddevice. One method is an Automated Cellular Expansion (ACE) system,which is a series of commercially available or custom fabricatedcomponents linked together to form a cell growth platform in which cellscan be expanded without human intervention. Cells are expanded in a celltower, consisting of a stack of disks capable of supportinganchorage-dependent cell attachment. The system automatically circulatesmedia and performs trypsiziation for harvest upon completion of the cellexpansion stage.

Alternatively, the ACE system can be a scaled down, single lot unitversion comprised of a disposable component that consists of cell growthsurface, delivery tubing, media and reagents, and a permanent base thathouses mechanics and computer processing capabilities forheating/cooling, media transfer and execution of the automatedprogramming cycle.

Upon receipt, each sterile irradiated ACE disposable unit will beunwrapped from its packaging and loaded with media and reagents byhanging pre-filled bags and connecting the bags to the existing tubingvia aseptic connectors. The process continues as follows:

Inside a biological safety cabinet (BSC), a suspension of cells from abiopsy that has been enzymatically digested is introduced into the“pre-growth chamber” (small unit on top of the cell tower), which isalready filled with Initiation Growth Media containing antibiotics. Fromthe BSC, the disposable would be transferred to the permanent ACE unitalready in place.

After approximately three days, the cells within the pre-growth chamberare trypsinized and introduced into the cell tower itself, which ispre-filled with Complete Growth Media. Here, the “bubbling action”caused by CO₂ injection force the media to circulate at such a rate thatthe cells spiral downward and settle on the surface of the discs in anevenly distributed manner.

For approximately seven days, the cells are allowed to multiply. At thistime, confluence will be checked (method unknown at time of writing) toverify that culture is growing. Also at this time, the CGM will bereplaced with fresh CGM. CGM is replaced every seven days for three tofour weeks. At the end of the culture period, the confluence is checkedonce more to verify that there is sufficient growth to possibly yieldthe desired quantity of cells for the intended treatment.

If the culture is sufficiently confluent, it is harvested. The spentmedia (supernatant) is drained from the vessel; PBS is pumped into thevessel (to wash the media, FBS from the cells) and drained almostimmediately; trypsin-EDTA is pumped into the vessel to detach the cellsfrom the growth surface; the trypsin/cell mixture is drained from thevessel and enter the spin separator; cryopreservative is pumped into thevessel to rinse any residual cells from the surface of the discs, and besent to the spin separator as well; the spin separator collects thecells and then evenly resuspend the cells in the shipping/injectionmedium; from the spin separator, the cells will be sent through aninline automated cell counting device or a sample collected for cellcount and viability testing via laboratory analyses. Once a specificnumber of cells has been counted and the proper cell concentration hasbeen reached, the harvested cells are delivered to a collection vialthat can be removed to aliquot the samples for cryogenic freezing.

Alternatively, automated robotic systems may be used to perform cellfeeding, passaging, and harvesting for the entire length or a portion ofthe process. Cells can be introduced into the robotic device directlyafter digest and seed into the T-175 flask (or alternative). The devicemay have the capacity to incubate cells, perform cell count andviability analysis and perform feeds and transfers to larger culturevessels. The system may also have a computerized cataloging function totrack individual lots. Existing technologies or customized systems maybe used for the robotic option, such as the products obtained from TheAutomation Partnership (TAP).

C. Preparation of Cell Suspension

At the completion of culture expansion, the cells are harvested andwashed, then formulated to contain 1.0-2.7×10⁷ cells/mL, with a targetof 2.2×10⁷ cells/mL. Alternatively, the target can be adjusted withinthe formulation range to accommodate different indication doses. Thedrug substance consists of a population of viable, autologous humanfibroblast cells suspended in a cryopreservation medium consisting ofIscove's Modified Dulbecco's Medium (IMDM) and Profreeze-CDM™ (Lonza,Walkerville, Md.) plus 7.5% dimethyl sulfoxide (DMSO). Alternatively, alower DMSO concentration may be used in place of 7.5%. Alternatively,CryoStor™ CS5 or CryoStor™ CS10 (BioLife Solutions, Bothell, Wash.) maybe used in place of IMDM/Profreeze/DMSO.

After completion of the controlled rate freezing step, Bulk DrugSubstance vials are transferred to a cryogenic freezer for storage inthe vapor phase. After cryogenic freezing, the Drug Substance issubmitted for Quality Control testing. Drug Substance specificationsalso include cell count and cell viability testing performed prior tocryopreservation and performed again for Drug Substance—Cryovial.Viability of the cells must be 85% or higher for product release. Cellcount and viability are conducted using an automated cell countingsystem (Guava Technologies), which utilizes a combination of permeableand impermeable fluorescent, DNA-intercalating dyes for the detectionand differentiation of live and dead cells. Alternatively, a manual cellcounting assay employing the trypan blue exclusion method may be used inplace of the automated cell method above or other automated cellcounting systems may be used to perform the cell count and viabilitymethod, including Cedex (Roche Innovatis A G, Bielefield, Germany),ViaCell™ (Beckman Coulter, Brea, Calif.), NuceloCounter™ (New BrunswickScientific, Edison, N.J.), Countless® (Invitrogen, division of LifeTechnologies. Carlsbad, Calif.), or Cellometer® (Nexcelom Biosciences,Lawrence, Mass.). Drug Substance—Cryovial samples must meet a cell countspecification of 1.0-2.7×10⁷ cells/mL prior to release. Sterility andendotoxin testing are also conducted during release testing.

In addition to cell count and viability, purity/identity of the DrugSubstance is performed and must confirm the suspension contains 98% ormore fibroblasts. The usual cell contaminants include keratinocytes. Thepurity/identify assay employs fluorescent-tagged antibodies against CD90and CD104 (cell surface markers for fibroblast and keratinocyte cells,respectively) to quantify the percent purity of a fibroblast cellpopulation. CD90 (Thy-1) is a 35 kDa cell-surface glycoprotein.Antibodies against CD90 protein have been shown to exhibit highspecificity to human fibroblast cells. CD104, integrin β4 chain, is a205 kDa transmembrane glycoprotein which associates with integrin α6chain (CD49f) to form the α6/β4 complex. This complex has been shown toact as a molecular marker for keratinocyte cells (Adams and Watt 1991).Antibodies to CD104 protein bind to 100% of human keratinocyte cells.Cell count and viability is determined by incubating the samples withViacount Dye Reagent and analyzing samples using the Guava PCA system.The reagent is composed of two dyes, a membrane-permeable dye whichstains all nucleated cells, and a membrane-impermeable dye which stainsonly damaged or dying cells. The use of this dye combination enables theGuava PCA system to estimate the total number of cells present in thesample, and to determine which cells are viable, apoptotic, or dead.

D. Dosage Units

Drug Substance—Cryovial used to prepare the final dosage unit consistsof fibroblasts that are harvested from the final culture vessel,formulated to the desired cell concentration and cryopreserved incryovials. Drug Substance-Cryovial is stored in a cryopreservationmedium consisting of IMDM and Profreeze™ plus 7.5% DMSO to a target of2.2×10⁷ cells/mL. After exposure to a controlled rate freezing cycle,the cryovialed Drug Substance is stored frozen in the vapor phase of aliquid nitrogen freezer.

Harvested cells are pooled, formulated in a cryopreservation media thatincludes Profreeze, DMSO and IMDM media, aliquoted into cryovials andstored frozen in liquid nitrogen as the Drug Substance—Cryovial materialvia controlled rate freezing.

The caps and vials are radiation sterilized and received sterile fromthe manufacturer. The required volume of bulk material needed fortreatment is removed from frozen storage, thawed, and pooled. The cellsare washed with 4× bulk volume of PBS and centrifuged at 150×g for 10minutes (5±3° C.). This is followed by a wash with 4× bulk volume ofDMEM by resuspension and centrifugation at 150×g for 10 minutes (5±3°C.). The washed cells are resuspended in DMEM without phenol red to atarget concentration of 1.0-2.0×10⁷ cells/mL. Alternatively, the second4× wash and final resuspension can be performed with Hypothermosol®-FRS(BioLife Solutions, Bothell. WA). The final sterile cryovial containersare then manually filled in a Biological Safety Cabinet to a volume of1.2 mL/container. The Drug Product—Injection is stored at 2-8° C. untilshipment in a 2-8° C. refrigerated shipper to the administration site.

Alternatively, Drug Substance vials can be removed from cryogenicstorage and shipped directly to the administration site for dilution andadministration. In the direct injection concept, the cells are harvestedand prepared for cryopreservation at a higher cell concentration(3.0-4.0×10⁷ cell/mL as compared to the current target of 2.2×10⁷cells/mL). When an injection is pending, the frozen vial will be shippedto the study site on dry ice or in a liquid nitrogen dewar. Theadministration site thaws the vial by hand or with a heat block, andperforms a 1:1 ratio dilution of the frozen cells at the study siteusing a typical injection diluent such as bacteriostatic water, sterilewater, sodium chloride, or phosphate buffered saline. Alternatively,DMEM may be used as the diluent. This concept eliminates the need towash and prepare a fresh suspension of the injection for overnightshipment to the study site.

Alternatively, cells freshly harvested from flasks or cells stacks canbe adjusted to a target concentration of 1.0-2.0×10⁷ cells/mL in DMEM,undergo all Bulk Harvest and Drug Substance—Cyrovial testing describedabove and shipped fresh overnight to the administration site in a 2-8°C. refrigerated shipper as the final injection product. In thisscenario, sterility and mycoplasma testing may be performed upstreamfrom the harvest to allow time for results prior to shipment.

II. Methods of Administration

A. Preparation of Dosage Units

A suspension of each patient's own living autologous fibroblastsformulated in Dulbecco's Modified Eagle's Medium (DMEM) without phenolred is supplied as two 2 mL vials with each vial containing 1.2 mL offibroblasts at 1.0-2.0×10⁷ cells/mL. A dose range from 1.1-5.8×10⁷cells/mL injection has been used successfully.

B. Administration of Dosage Units

Vials are warmed to room temperature and gently inverted to resuspendthe settled cells. The cellular suspension is withdrawn from thecontainer using a small unit syringe fitted with a detachable needle orwith a fixed needle. A 27 gauge, 1½ inch needle is required forinjection of the product into the vocal folds. However, a syringe with alarger bore (18 or 21-gauge) detachable needle may be used to aid inwithdrawing the product from the container. Once withdrawn, thelarger-gauge needle can be switched out with a 27-gauge needle and theproduct administered. Product administration is accomplished using adistal chip flexible fiberoptic laryngoscope to visualize the vocalfolds, then advancing the injection needle into the larynx using thetrans-cricothyroid membrane technique. The product is then layered inthe vocal fold subepithelial layer (in the lamina propria compartment).

C. Conditions to be Treated

Autologous fibroblasts are injected into the lamina propria layer totreat vocal fold scarring. In the preferred treatment regime, threetreatment doses of 1-2×10⁷ cells/mL are injected into the superficiallamina propria layer of each scarred vocal fold one to eight, preferablyfour weeks apart.

EXAMPLE 1 A Phase I Clinical Trial to Determine the Safety and Efficacyof Autologous Fibroblast Cellular Injection Therapy for Treatment ofVocal Fold Scarring

Objectives: The extracellular matrix and cellular components of thelamina propria layer is lost or altered in vocal fold scars. The goal ofthis study was to assess the safety and effectiveness of autologousfibroblasts injection to the lamina propria layer to treat vocal foldscarring.

Results: Five patients were enrolled into the study with vocal foldscars that ranged from isolated lamina propria loss to full thicknessscarring that included the epithelium. All subjects received alltreatments and were followed up to 12 months. Assessment of safetyendpoints showed that the only adverse events were temporary otalgiaduring treatment injection in the two patients with full thickness coverscars. There was no other morbidity or mortality. There were nolaboratory abnormalities or other untoward events that were consideredrelated to the study treatment. The primary efficacy endpoint of changefrom baseline in the mucosal wave grade showed a sustained improvementfrom week 8 through the Month 12 visit. A trend for sustainedimprovements through month 12 was also noted for the VHI, VAS, and VoiceQuality Questionnaire. No trends for improvement were noted forHarmonic-to-Noise ratio and maximal phonation time.

Conclusions: This study showed that injection of autologous fibroblastsinto the scarred vocal fold lamina propria layer is safe. Sustainedtrends for improved outcome were supported by 12-month data for MucosalWave Grade, VHI, VAS Assessment of Vocal Fold Improvement, and VoiceQuality Questionnaire.

Methods 1. Overview of Experimental Design

Five subjects with vocal fold scarring who met the inclusion andexclusion criteria were selected from a voice clinic for enrollment.After enrollment into the study, punch biopsies of buccal mucosa wereobtained. The biopsy samples are sent to a commercial laboratory forlaboratory expansion of the fibroblast cell population. After adequateexpansion of the cells, subjects received percutaneous injections of theexpanded autologous fibroblasts into the subepithelial layer (laminapropria compartment) of the scarred vocal folds. Subjects receivedtreatment to one or both vocal folds, depending upon whether unilateralor bilateral scarring was present. Only one vocal fold was treated ateach treatment session, alternating to the opposite vocal fold (if beingtreated bilaterally) at the next treatment session. Each scarred vocalfold was treated a total of three times (total of three treatmentsessions for unilateral vocal fold scarring and six treatment sessionsfor bilateral vocal fold scarring).

Safety and efficacy assessments were performed at various time intervalsas detailed below. Follow-up examinations were performed at 3, 4, 8, and12 months following the first injection. Efficacy assessments includedlaryngeal videostroboscopy, voice recording for acoustic analysis ofHarmonic-to-Noise ratio, and completion of questionnaires including theVoice Handicap Index (VHI), visual analog scale (VAS) instrument, and avoice questionnaire.

II. Subjects

Five adults 18 years of age or older were selected for the study. Thenumber of subjects for the study and the plan to inject only one vocalfold per treatment session was decided after careful discussion of theresearch protocol and the safety of the procedure with representativesof the Federal Drug Administration, who approved the research protocoland the Investigational New Drug Application for this study.

(a) Inclusion Criteria:

(1) At least 18 years of age.(2) Presence of unilateral or bilateral vocal fold scarring as diagnosedby medical history and videostroboscopic examination of the larynx.(3) Grade 1-2 mucosal waves (see below for grading) as determined byvideostroboscopy.(4) Failed any one or more alternative treatments including but notlimited to anti-reflux regimen, speech therapy, or vocal fold injectionlaryngoplasty at least 4 months prior to screening.(5) Self-reported that their voice quality was a major handicap.(6) If female and capable of bearing children agreed to use a medicallyacceptable means of birth control during the study and tested negativeon a pregnancy test prior to the administration of first treatment.(7) Willing and able to follow study procedures and instructions.(b) Exclusion Criteria—Subjects were Excluded from the Study forPresence of any of the Following:(1) An active smoker.(2) Had an upper respiratory infection at baseline (subject could berescheduled).(3) Was already participating, or had within 30 days prior to enrollmentparticipated in another clinical trial involving therapeuticintervention.(4) Had other concurrent laryngeal pathology including lesions thatwould require removal.

III. Study Procedures—Subjects Underwent the Following Study RelatedProcedures (a) Buccal Mucosal Biopsy

After enrollment into the study, 3-mm punch biopsies were taken from thebuccal mucosa of the subject under clean conditions. Three biopsies weretaken and the tissue was sent via overnight courier to a commercial cellproduction facility (Isolagen Technologies, Inc., Exton, Pa.) for growthand expansion of fibroblast cells. The biopsy was placed in a vial withmedia that maintained viability of the specimen for at least 48 hoursand then shipped immediately in controlled ambient temperaturepackaging. Buccal mucosa rather than skin biopsy was chosen as thesource for autologous fibroblasts because the collagen productionprofile of mucosa parallels that of the vocal fold fibroblasts ascompared to dermal fibroblasts (Krishna et al. Otolaryngol Head NeckSurg 2006; 135:937-945). In addition, buccal mucosa is easier to biopsy,leaves no visible external scar, and heals very well without sutureclosure of the biopsy defect.

(b) Laboratory Expansion of Autologous Fibroblasts

Once received by the laboratory, the buccal mucosal biopsy was digestedusing a Liberase and seeded into a culture flask with Iscove's ModifiedDulbecco's Medium (IM DM) and GA, which was used throughout the cellculture protocol. When a fibroblast cellular monolayer was establishedit was digested with trypsin to liberate fibroblasts. The fibroblastswere then cultured in larger flasks. When cells reached the establishedconfluence specification, they were treated with trypsin and transferredto a larger flask with IMDM. When adequate cellular expansion hadoccurred (between 4th to 6th passages), screening and in-processcontrols for bacterial, fungal and mycoplasma contamination, cellmorphology, and confluence of monolayers were completed, and theexpanded autologous fibroblasts were harvested into IMDM. The harvestedcells suspension was mixed with a cryopreservant containing a lowconcentration of dimethylsulfoxide (DMSO) for cryopreservation. Thecryopreserved cells could be recultured or simply washed and prepped toremove DMSO and replace the remaining medium with DMEM prior to shippingfor injection. Cell counts, viability, endotoxin testing, Gram stain,and a final sterility test were then performed on each cell suspension.To complete the injection preparation process, 1.2 mL (1.0-2.0×10⁷cells) of the final product was filled in a 2 mL cryovial, packaged inan insulated vial container and shipped at 2-8° C. to the treatingfacility for vocal fold injection. Upon receipt the vial was stored in arefrigerator at 4° C. until ready for injection.

(c) Preparation of Cells for Injection

All vials were received by the study site on the morning of injectionand were prepared in the same manner. Shortly before the administrationof autologous fibroblasts, the subject's treatment vial was taken out ofthe refrigerator and allowed to warm to room temperature. The cells werere-suspended by gently inverting the injection vial three times beforeaseptically drawing the vial's contents into a sterile syringe with an18-gauge needle. Before administration of autologous cells, the 8-gaugeneedle was replaced with a 27-gauge, 1½-inch needle.

(d) Vocal Fold Injection

All injections were performed by the investigator who routinely performsin-office injection laryngoplasty. Subject was seated comfortably on anexamination chair with the neck slightly extended to expose laryngeallandmarks. The nasal cavity was decongested with neosynephrine, and thenasal cavity and larynx are anesthetized with topical 4% lidocainespray. The neck skin was not routinely anesthetized unless requested bythe subject or it was felt that injection would go smoother if the neckskin was anesthetized. A distal chip flexible fiberoptic laryngoscope(VNL 1170K, KayPentax, Lincoln Park, N.J.) connected to a video monitorwas passed via the nostril into the nasal cavity and advanced to thehypopharynx until the vocal folds were visualized. The needle entry siteon the anterior neck skin was prepped with alcohol swab. The injectionneedle was advanced into the larynx using the trans-cricothyroidmembrane technique. While visualizing on the video monitor, autologouscells were layered in the vocal fold subepithelial layer (in the laminapropria compartment). Appropriate placement was confirmed visually asexpansion of the epithelial layer due to superficial injection. Subjectswere observed in the clinic for one hour prior to discharge.Prophylactic antibiotics were not prescribed.

(e) Other Therapy

No other vocal fold therapies were permitted in conjunction with thestudy treatments and for the duration of follow-up through 12 months.

IV. Assessment of Safety

Assessments of subject safety included strict maintenance of case reportforms (CRFs), tabulations of the incidence of adverse events (AEs), andanalysis of changes from baseline in laboratory values.

V. Assessment of Efficacy (a) Summary of Efficacy Endpoints

The primary efficacy endpoint was the absolute change from baseline inmucosal wave grade using videostroboscopy (described below) on eachtreated vocal fold. Efficacy endpoints were collected at week 4, week 8,month 3, month 4, month 8, and month 12 after first injection treatment.

The secondary efficacy endpoints of this feasibility study were thefollowing outcomes measures (described below):

(1) Subject's impression of improvement in voice quality using a VisualAnalog Scale (VAS)(2) Subject's impression of improvement in voice quality usingquestionnaire(3) Absolute change from baseline in the Harmonic-to-Noise ratio (dB) asmeasured from acoustic analysis of voice(4) Absolute change from baseline in the Voice Handicap Index (VHI)(5) Absolute change from baseline in maximum phonation time (MPT)

(b) Clinical Assessments (1) Videostroboscopy:

The primary effect assessment was the absolute change from baseline inmucosal wave grade using videostroboscopy. The investigator performedall videostroboscopic procedures. Both a 70 degree rigid endoscope and adistal chip flexible fiberoptic endoscope were used initially forvideostroboscopy. It was determined that the mucosal wave grade in eachpatient was the same for both endoscopes and therefore the rigidendoscope was used for most of the study period due to its superioroptical quality. During the videostroboscopic procedure the endoscopeattached to a miniature video camera was used to visualize the larynxunder stroboscopic light source. The subject was asked to phonate asustained vowel “e” and the vocal fold vibration was recorded digitallyon a computer for analysis. The recordings were reviewed later by alaryngologist and an experienced speech pathologist and a consensus wasgenerated on the mucosal wave grade. Mucosal waves were graded asfollows: 1=absent; 2=limited to the most medial edge of the vocal folds;3=present laterally up to ¼ of the width of the vocal folds; 4=presentup to but less than ½ the width of the vocal folds; 5=present at morethan ½ the width of the vocal folds (normal).

(2) Voice Recording for Acoustic Analysis

A sample of voice (a continuous vowel sound /a/) was recorded anddigitized to measure acoustic parameters of voice using computersoftware. Acoustic parameters include Harmonic-to-Noise ratio, jitter,and shimmer. Only Harmonic-to-Noise ratio was calculated because theacoustic measures of jitter and shimmer have not been found to be robustin perception of voice quality. In a pathologic voice, Harmonic-to-Noiseratio is decreased. Another acoustic parameter measured was maximalphonation time (MPT). MPT was defined as the length of time in seconds asubject was able to phonate a sustained vowel in one single deep breathand was averaged over three attempts. Glottic incompetence is typicallypresent in vocal fold scars and sometimes leads to excessive airflowloss from decreased glottic resistance if the glottic gap is large.

(3) Voice Handicap Index (VHI)

Subjects completed a Voice Handicap Index (VHI) survey, which is a30-item test, developed by Jacobson and colleagues with 10 items inthree subscales: emotional, physical, and functional. Each item isanswered on a 5-point scale from “0”, indicating the subject never feltthis about the voice problem to “4”, where the subject always felt thisto be the case. Thus highest VHI rating per subscale is 40 points andfor the entire survey 120 points. Each subscale has been found to besignificantly different if it changes by eight points, whereas the totalVHI score was found to be significantly different if it changes by 18points.

(4) Subject's VAS Assessment of Vocal Fold Impromvement

Subjects were asked to rate the vocal fold improvement using a VisualAnalog Scale (VAS). The VAS will range from “Worst Possible Change FromBaseline” on the left side to “Most Possible Improvement From Baseline”on the right side. The VAS at each assessment were measured and given ascore from −50 to 50.

(5) Subject's Impression of Voice Quality Questionnaire

Subjects were asked two questions to assess the subject's satisfactionwith treatment with the following answer choices:

1. How has your voice quality changed since baseline?

a. Improved b. No Change c. Worsened

2. Do you consider the treatment a success?

a. Yes b. No

At each assessment, the proportion of subjects that reported eachresponse was tabulated, and 95% confidence intervals calculated.

VI. Statistical Considerations

Version 8.0 or higher of the SASV statistical software package was usedto perform all statistical analyses. When there were missing valuesstatistical evaluations were performed using non-missing values.

Sample size: There was insufficient clinical experience on how this newprocedure would affect vocal fold scarring to estimate sample size. Thedecision to limit sample size to five was selected after discussion withthe FDA. The sample size is, however, typical of an exploratory, earlyphase study in which the objective is to investigate the feasibility oftreatment administration

Safety Endpoints: Assessments of subject safety included tabulations ofthe incidence of adverse events and analysis of the percentage changefrom baseline in laboratory measurements. Laboratory measurements wereinvestigated by calculating the percentage changes from baseline anddetermining if a statistically significant change from baseline wasdetected using a paired t-test. Each laboratory result was also flaggedas low (L), high (H) and normal (N) based on the lab normal range.Adverse events with an onset during the course of study were recordedand tabulated.

Efficacy Endpoints: The primary effect endpoint analysis of change frombaseline in mucosal wave grade using videostroboscopy on each treatedvocal fold was assessed with Wilcoxon Signed-Rank test. Secondaryeffects analysis of changes in Maximal Phonation Time andHarmonic-to-Noise ratio was tested using the Wilcoxon Signed-Rank test.The VAS at each assessment was measured and given a score from −50 to50. Summary statistics on this change from baseline was provided andtested for differences from zero using the Wilcoxon Signed-Rank test.The Subject's Impression of Voice Quality Questionnaire was evaluated bytabulating the proportion of subjects that report each response, and 95%confidence intervals calculated. The VHI findings were reportedindividually for each subscale and for the entire survey. Each subscalehas been found to be significantly different if it changes by eightpoints, whereas the total VHI score has been found to be significantlydifferent if it changes by 18 points.

Results Subjects:

Five subjects who were evaluated in a voice clinic for dysphonia and metinclusion and exclusion criteria were enrolled and completed the study.Three (60%) were male and two (40%) were female. The average age was 55years, with a range of 35 to 68 years and a median age of 58 years. Allfive were Caucasian. Prior treatments included anti-reflux medication (5of 5, 100%), speech therapy (5 of 5, 100%), injection augmentationlaryngoplasty with collagen (3 of 5, 60%), and other (2 of 5, 40%). Allwere in good health and none had a history of tobacco use. Four patientswere taking concomitant medications (analgesics, antacids,anti-inflammatory medications, etc.) but none of these were expected toaffect the analysis of safety or efficacy of treatment. All fivesubjects received all scheduled treatments with autologous fibroblasts.The subjects represented the spectrum of vocal fold scars from isolatedlamina propria loss to full thickness cover defect as follows:

Subject 1 was a 35 years old male who noted onset of dysphonia after atonsillectomy five years prior to enrollment in this study, Otherassociated medical disorders included possible gastroesophageal refluxdisease (GERD). Prior therapies for dysphonia included anti-refluxregimen and voice therapy. Videostroboscopic exam at enrollment revealedgrade 2 mucosal waves bilaterally and incomplete glottic closure. Therewere no defects on the epithelium and the vocal fold scar wascategorized as isolated lamina propria loss with intact epitheliallayer.

Subject 2 was a 47 years old male who had onset of dysphonia six yearsprior to enrollment in this study. He was found to have bowing of vocalfolds and underwent several collagen injection laryngoplasties withoutimprovement. Additional therapies included anti-reflux regimen and voicetherapy. Videostroboscopic exam at enrollment revealed grade 1 mucosalwaves on the left and grade 2 mucosal waves on the right vocal fold andincomplete glottic closure. There were no defects on the epithelium andthe vocal fold scar was categorized as isolated lamina propria loss withintact epithelial layer.

Subject 3 was a 66 years old female who had onset of dysphonia afterbeing assaulted to the neck area 39 years prior to enrollment in thisstudy. She then developed supraglottic squamous cell carcinoma 33 yearsprior to enrollment and was treated with external beam radiationtherapy. She further suffered from right vocal fold paralysis after acarotid endarterectomy 13 years prior to enrollment. Prior therapies forher voice disorder included anti-reflux regimen, voice therapy,injection laryngoplasty with collagen, and fat injection.Videostroboscopic exam at enrollment revealed grade 1 mucosal wavesbilaterally and incomplete glottic closure. The epithelium appearedscarred and bilaterally and the vocal fold scar was categorized as fullthickness scar of the vocal fold involving both the lamina propria andepithelial layers.

Subject 4 was a 62 years old male who underwent direct laryngoscopy withexcision of right vocal fold nodule 30 years prior to enrollment in thisstudy with resultant long-term dysphonia after surgery. Prior therapiesfor his voice disorder included multiple collagen injectionlarygoplasties 3 to 7 years prior to enrollment. Four years prior toenrollment he had superficial collagen injection removed under directlaryngoscopy and three years prior he had bilateral type 1 thyroplastieswith silastic implants performed. Videostroboscopic exam at enrollmentrevealed a right vocal fold sulcus (linear defect on the vocal foldsurface throughout the length of the membranous vocal fold) with grade 1mucosal wave, and an intact left vocal fold with grade 4 mucosal waves.The vocal fold scar was categorized as unilateral sulcus type. Subject 4was the only enrollee to receive unilateral vocal fold treatment in thisstudy.

Subject 5 was a 68 years old female who underwent laser ablation ofReinke's edema four years prior to enrollment. Her dysphonia ensued.Subsequent treatments included three separate treatments with fatinjections. She responded to the first two injections but not to thethird. Six months prior to enrollment she underwent injectionlaryngoplasty with calcium hydroxylapatite. She continued to be severelydysphonic. Videostroboscopic exam at enrollment revealed grade 1 mucosalwaves bilaterally. The epithelium appeared scarred bilaterally and thevocal fold scar was categorized as full thickness scar of the vocal foldinvolving both the lamina propria and epithelial layers.

Buccal Biopsy and Cellular Expansion:

All five subjects underwent buccal mucosal biopsy without complicationsand all biopsy sites rapidly healed by secondary intention. An averageof 0.122 days (range 63-196 days) was required from biopsy to treatment.One patient required a second biopsy due to bacterial contaminationdetected late in the fibroblast expansion process. Cell expansion wasotherwise unremarkable and cell specifications were reached for allsubjects. Cell viability at injection was above specification (>85%) inall subjects (actual range 86%-96%). Cell concentration was withinspecification (1.0-2.0×10⁷ cell/mL) in all injections (actual range 1.1to 2.0×10⁷ cells/mL). Gram stains were performed in samples from eachtreatment vial prior to packaging and were negative. An aliquot fromeach injection vial was also used for culture and no microbiologiccontaminants, including mycoplasma, were cultured from the monitoredsamples. Mycoplasma was tested at the Bulk Harvest stage, not in thefinal injection. Sterility test was performed for each vial filled, butreleased using Gram stain. The sterility test was not finalized untilafter injection.

Safety Endpoints:

All five subjects received study treatment of 1-2×10⁷ cells/mL. Eachscarred vocal fold received three treatments at four week intervals. Onesubject (Subject 4) had unilateral vocal fold scarring and received atotal of three injections. The other four subjects had bilateral vocalfold scarring and received a total of six injections each. Thus patientswith bilateral vocal fold scarring were seen at 2-week intervals whereasonly one vocal fold was injected per visit. Of the total 27 treatmentinjections performed, the full treatment dose of 1.0 ml was injected in24 instances (89%). In the other three instances 0.85 ml (Subject 1,Treatment #1), 0.5 ml (Subject 5, Treatment #6), and 0.3 ml (Subject 3,Treatment #6) were injected. In the first instance, less than 1.0 mL wasinjected because a small amount was lost during aspiration of cells fromthe treatment vial, while in the latter two instances the treatmentinjections were terminated prematurely due to complaints of severeotalgia during the injection.

Subjects were observed for one hour after each treatment injection.Repeat endoscopy was performed the day after the first treatment.Subjects were contacted by telephone on days 2 through 4 after treatmentto check on the airway status and other constitutional symptoms such asfever, chills, neck swelling, or other new symptoms. There were noadverse reactions noted by the investigator or reported by the subjects.

During each treatment and follow-up visits adverse events (AEs) wereactively sought and collected. Two subjects experienced a total of 12AEs considered related to treatment. These were mild to severe otalgiasuffered during treatment in subjects 3 and 5. These were subjectscategorized as having extensive full thickness vocal fold scars of thecover layer, involving scarring of the epithelial layer as well aslamina propria loss. The otalgia was considered definitely related tostudy treatment as it was experienced by these subjects as they werereceiving the injection treatment, and the pain symptoms correlatedtemporally with the moment the cells being injected in the laminapropria compartment were dissecting the epithelial layer off from themuscle layer. The otalgia was mild to moderate in 10 instances andsevere in 2 instances. As mentioned earlier, in the latter two instancesthe treatment was terminated prematurely prior to injection of the full1.0 mL dose. The otalgia resolved rapidly after the injection wasterminated and was absent by the time of discharge from the clinic. Theother three subjects had no otalgia complaints. There were no deaths orserious adverse events. No subject was discontinued from the study dueto AEs.

Laboratory assessments were analyzed by flagging each laboratory valuelow (L), high (H), and normal (N) based on the investigational site labnormal range. The percentage change from baseline was also calculatedand the Wilcoxon Signed-Rank test was used to determine if astatistically significant change from baseline was present. There wereno clinically significant changes in laboratory parameters. In fact allthe laboratory parameters were within normal in all subjects at allassessment time periods. There were no clinically significant changes invital signs (respiration rate, heart rate, temperature, and bloodpressure) from baseline during the course of the study (WilcoxonSigned-Rank Test).

Primary Efficacy Endpoint: Mucosal Wave Grade:

The primary efficacy endpoint assessed in this study was the change frombaseline in mucosal wave grade using videostroboscopy of each treatedvocal fold. Table 1 lists each subject by scar type and provides mucosalwave grade for each subject at various assessment intervals. Note thatnot all five subjects completed the assessment for each visit and allvalues were calculated based on non-missing values. A positive change inmucosal wave grade from baseline indicated improvement. The mean changefrom baseline in mucosal wave grade showed improvement at week 8, andsignificantly at months 3, 4, and 12. This demonstrates sustainedimprovement in mucosal wave starting at week eight and continuing up tothe end of the study assessment at month 12.

A closer review of the mucosal wave data reveals that the two subjectswith the isolated lamina propria loss (Subjects 1 and 2) and the subjectwith sulcus scar (Subject 4) improved most with treatment. The twosubjects with full thickness cover scarring (Subjects 3 and 5) hadessentially no improvement; subject 3 had no improvement in mucosalgrade at all assessment time intervals, and subject 5 continued to havesevere scarring throughout the study period (mucosal wave grade 1-2)until the 12 month interval assessment when the left vocal fold appearedto have slightly improved mucosal wave (mucosal wave grade 3).

Review of videostroboscopic recording in subject 4 revealed thatalthough the mucosal waves improved, the physical appearance of thesulcus (linear defect on the vocal fold surface) remained unchangedthroughout the study period. Lower scores indicate improved perceptionof the impact of the voice disorder by the subject. The total score isgiven followed by the change (in parenthesis) compared to baseline VHIscore at day 0. A change of 18 points from Day 0 is consideredsignificantly different (bolded).

TABLE 1 Demographic, Past Vocal History, and Laryngoscopic FindingsEtiology or MW Subject Associated Failed grade Status of GlotticNo/Sex/Age Disorders Duration Prior Rx Right Left Epithelium Gap 1/35/MTonsillectomy  5 years PPI, Voice Rx 2 2 intact Yes GERD 2/47/M Bowing 6 years PPI, Voice Rx, 2 1 Intact Yes IL 3/66/F SCCA/XRT 33 years PPI,Voice Rx, 1 1 Scarred Yes Right VFP 13 years IL, Fat Injection 4/62/MCold MDL 30 years PPI, Voice Rx, 1 4 Sulcus Yes for nodule IL, Type 1Thyroplasty 5/68/F Laser MDL  4 years PPI, Voice Rx, 1 1 Scarred Yes forIL edema Fat Injection GERD = Gastro Esophageal Reflux Disease, SCCA =Squamous Cell Carcinoma, XRT = Radiation Therapy, VFP = Vocal FoldParalysis, MDL = Microsuspension Direct Laryngoscopy with excision oflesion, PPI = Proton Pump inhibitor, Voice Rx = Voice Therapy, IL =Injection Laryngoplasty

Secondary Efficacy Endpoints: Harmonic-to-Noise Ratio:

The change from baseline in Harmonic-to-Noise ratio was examined for allsubjects at each assessment. Sounds that are considered noise generallyhave no harmonic structure. A smaller (or negative) value ofHarmonic-to-Noise ratio (dB) indicates more noisy voice signal and thusincreased vocal fold pathology. The absolute change from baseline inHarmonic-to-Noise ratio was calculated at each time point by subtractingthe Harmonic-to-Noise ratio at each respective assessment from theHarmonic-to-Noise ratio at baseline. These values were tested forstatistically significant differences from baseline using the WilcoxonSigned-Rank test. A positive absolute change from baseline in theHarmonic-to-Noise ratio indicates improved vocal fold pathology.

Table 2 provides summary information for subject assessment for VoiceRecording for Acoustic Analysis of Harmonic-to-Noise ratio. Theseresults suggest that most subjects experienced little change frombaseline in the voice recording for acoustic analysis of theHarmonic-to-Noise ratio after treatment. A closer examination of themeasurements shows a random distribution of this measure and likelymeans that this measure is not useful in this group of subjects.

TABLE 2 Mucosal Wave Grade at Various Intervals Vocal Month Subject ScarType Fold Day 0 Week 4 Week 8 Month 3 Month 4 Month 8 12 1 Lamina Left 2— 2 — 4 — 4 propria only Right 2 — 3 — 4 — 4 2 Lamina Left 1 1 3 5 4 — 5propria only Right 2 2 4 5 5 — 5 3 Lamina Left 1 1 1 1 1 1 1 propria &Right 1 1 1 1 1 1 1 epithelium 4 Sulcus Right 1 2 4 4 4 4 4 5 LaminaLeft 1 1 1 2 2 2 3 propria & Right 1 1 1 2 2 2 2 epithelium Mean MucosalWave Grade 1.3 1.3 2.2 2.9 3 2 3.22 Wilcoxon Signed Rank p-value 0.320.07 0.04 0.02 0.10 0.02 Mucosal Wave Grades Prior to and FollowingTreatment with autologous fibroblasts. Subjects 1, 2, 3, and 5 receivedsix treatments (3 to each vocal fold) starting Day 0 and completing atWeek 10 as described in Materials and Methods. Subject 4 received threetreatments with starting at Day 0 and completing at Week 8, Mucosal wavegrades were assigned by consensus between a laryngologist and anexperienced speech pathologist. Missing grades (—) indicate that thetest was not performed for that visit. Statistical significance of thechange from baseline was assessed with Wilcoxon Signed-Rank test. “ScarType” indicates the baseline assessment of scar (involvement of laminapropria only or lamina propria and epithelium).

Maximum Phonation Time:

The change from baseline in Maximum Phonation Time was examined for allsubjects at each assessment. A longer phonation time (sec) indicates animproved respiratory and laryngeal sound control. The absolute changefrom baseline in the Maximum Phonation Time was calculated at each timepoint by subtracting the baseline Maximum Phonation Time from theMaximum Phonation Time at each respective assessment. These values weretested for statistically significant differences from baseline using theWilcoxon Signed-Rank test. A positive absolute change from baseline inthe Maximum Phonation Time indicates improved vocal fold pathology.

Table 3 provides summary information for acoustic analysis of MaximumPhonation Time. Table 3 shows improvement from baseline beginning at theWeek 8 visit with a median change of 1.0. At Months 3 and 4, the MaximumPhonation Time showed further improvement, with median changes of 4.0and 2.3, respectively. However, by Month 8 there was a worsening frombaseline and at Month 12 only a minimal improvement from baseline wasseen. These results suggest a trend for improvement in vocal foldpathology at Months 3-4 that is not sustained.

TABLE 3 Voice Handicap Index VHI) Scores at Various Time Intervals bySubject Subject Day 0 Week 4 Week 8 Month 3 Month 4 Month 8 Month 12 1101  68 (−33) 75 (−26) 56 (−45) 56 (−45) 2 82 79 (−3) 84 (+2)  66 (−16)69 (−13) 67 (−15) 55 (−27) 3 79 75 (−4) 52 (−27) 55 (−24) 50 (−29) 51(−28) 50 (−29) 4 68 62 (−6) 41 (−27) 36 (−32) 41 (−27) 34 (−34) 30 (−38)5 89 98 (+9) 77 (−12) 79 (−10) 63 (−26) 58 (−31) 72 (−17) Total with 1/53/5 2/4 4/5 3/4 4/5 significant change

Voice Handicap Index (VHI):

Assessment of the VHI consisted of testing the absolute and percentagechange from baseline at each follow-up assessment. The VHI scores werecalculated for each of the three subscales and the overall total. Eachof these was tested for differences from baseline. Additionally, thepercentage change from baseline in VHI was calculated by subtracting thescore at the respective assessment from the score at baseline anddividing that difference by the baseline score and multiplying by 100%.These values were tested for statistically significant differences frombaseline using the Wilcoxon Signed-Rank test.

Note that higher VHI scores indicate a more severe perception of theimpact of the voice problem. The absolute change from baseline wascalculated by subtracting the score at the respective assessment fromthe score at baseline. Each subscale has been found to be significantlydifferent if it differed by eight points, whereas the total VHI scorehas been found to be significantly different if it differs by 18 points(21). FIG. 3 illustrates that most of the change in overall VHI scoreoccurred between weeks 8 and month 3 and that the change was maintainedfobr the rest of the study period.

Subject assessment for VHI—Part 1 (Functional) shows a positive absolutechange, which indicates improvement, beginning at the Week 4 visit andsustained through the Month 12 visit. Similar changes were indicated inthe percentage change from baseline. These results suggest a trend forimprovement in the VII—Part 1 (Functional) that is sustained throughMonth 12.

Subject assessment for VHI—Part 2 (Physical) showed similar results forthe VHI—Part 2 (Physical) as for the VHI—Part 1 (Functional). Sustainedimprovement from baseline was seen at Week 4 through Month 12.

The VHI—Part 3 (Emotional) showed similar results to both VHI—Part 1 and2. An improvement from baseline was seen at Week 8 that was sustainedthrough Month 12.

The VHI-Overall showed the same results as the VHI subparts.Improvements from baseline were seen at all study visits. These resultssuggest a trend for acute improvement that is sustained through 12months.

Subject's I/AS Assessment of Vocal Fold Improvement:

The subject was asked to rate the vocal fold improvement using a VAS atWeek 4, Week 8, and during all follow-up visits. The VAS ranged from“Worst Possible Change from Baseline” (score of −50) on the left side to“Most Possible Improvement from Baseline” (score of 50) on the rightside. Thus the VAS at each assessment was measured and given a scorefrom −50 to 50. These values were tested for statistically significantdifferences from baseline using the Wilcoxon Signed-Rank test. Apositive value indicates improvement.

Subject's VAS Assessment of Vocal Fold Improvement shows, beginning atWeek 8 subjects indicated improvement from baseline with a median changeof 17.0. At Months 3, 4, 8, and 12, subjects continued to indicateimprovement with median changes of 22.0, 18.0, 17.5, and 30.0respectively. At Month 4 one subject (subject #4) entered a negative VAS(score −28) and yet answered “improved” to the question “Has your voicechanged since baseline” and “Yes” to the question “Do you consider thetreatment a success?”, thus affecting the p-value significance trend atMonth 4. Nevertheless, these results suggest a trend for improvement inthe VAS Assessment for Vocal Fold Improvement that is sustained throughMonth 12.

Subject's Impression of Voice Quality Questionnaire:

At each assessment, the proportion of subjects that reported eachresponse was tabulated, and 95% confidence intervals calculated.Subject's Impression of Voice Quality Questionnaire for the question“How has your voice quality changed since baseline?” shows thatbeginning at Week 8, the majority of subjects indicated “Improved” forthe question. These results indicate that most subjects considered thattheir voice quality had improved since baseline, and that thisimprovement was sustained through the Month 12 visit. Subject'sImpression of Voice Quality Questionnaire for the question “Do youconsider the treatment a success?” shows that, beginning at Week 8, themajority of subjects indicated that the treatment was a success at eachvisit, most indicating “YES” for the question. These results indicatethat most subjects considered their treatment to be successful, and thatthis benefit was sustained through Month 12.

Discussion

The results show that autologous fibroblast therapy significantlyimproves mucosal waves in this cohort. Assessment of the primaryefficacy endpoint, the mucosal wave grade, showed an improvement at theweek 8 visit that was sustained until the end of the study (Month 12).While the small number of subjects in this Phase I trial limitedstatistical significance for many secondary measures, a trend towardsstatistical significance was achieved. The results for analysis of theVoice Handicap Index (all categories) showed sustained improvements frombaseline. The Voice Quality Questionnaire showed that the majority ofsubjects considered that their voice quality had improved, and that thisimprovement was sustained throughout Month 12. Acute improvement thatwas not sustained after Month 4 was observed for maximal phonation time.The assessment of Harmonic-to-Noise ratio showed no meaningful changesor trends. In addition, the most important goal, to show that autologousfibroblasts can be harvested and expanded in the laboratory and safelyre-injected into the subject, was achieved. The only adverse eventrelated to the study treatment was otalgia during vocal fold injection.It appears that subjects with full thickness scars are more susceptibleto otalgia and that in most instances the pain was tolerable enough sothat the procedure could be completed and the full treatment dose given.

Another important finding in this study is that subjects with fullthickness cover defects had minimal improvement of their mucosal wavesalthough improvement in other measures was noted. It is possible thatthe stroboscopic measure of mucosal wave grade does not yieldinformation that is obtained with other measures. For example, subjectsmay have perceived improved efficiency of phonation, or there may havebeen improvement in other factors that were not measured in this studythat lead to subjects' improved perception of voice. Finally, one mustalso consider the placebo effect of repeated injections. It appears thatthe when vocal fold scars are extensive and the regenerated epitheliumis also scarred and stiff replacement of the lamina propria layer aloneis unlikely to improve the mucosal wave, which requires a pliableepithelial layer as well. These defects may be better treated with fullthickness cover replacement.

We claim:
 1. A method for treatment of vocal fold scarring,presbylaryngis and vocal cord augmentation in a human in need thereofcomprising injecting into the vocal cord or fold a dosage formulationcomprising between 1.0 and 2.7×10⁷ cells/mL autologous human fibroblastcells or precursors thereof, at least 85% of which are viable.
 2. Themethod of claim 1 wherein between one and two ml of the cells isadministered.
 3. The method of claim 1 wherein the cells are layered inthe vocal fold subepithelial layer.
 4. The method of claim 1 furthercomprising providing local anesthetic to the nasal cavity or larynx. 5.The method of claim 1 further comprising visualizing the site ofinjection using a fiberoptic laryngoscope connected to a video monitor.6. The method of claim 1 further comprising obtaining fibroblast cellsfrom the buccal mucosa of a human and culturing and purifying the cellsprior to administration.
 7. The method of claim 1 wherein 98% or more ofthe cells are fibroblasts.
 8. The method of claim 1 wherein one, two,three or four treatment doses of 1-2×10⁷ cells/mL were injected into thesuperficial lamina propria layer of each scarred vocal fold.
 9. Themethod of claim 8 wherein the doses are administered one to eight weeksapart.